Polyketides including tacrolimus (FK506), rapamycin, and ascomycin (FK520) are natural products having a complex structure, produced by microorganisms, and more than 10,000 compounds are known. Mainly anticancer agents, antibiotics, antihypertensive agents, immunosuppressive agent or the like have been developed from these compounds, and account for approximately 50% of natural product-derived drugs currently used. Tacrolimus, rapamycin and ascomycin have a similar structure to inhibit T cell activation both in vitro as well as in vivo. It was reported that these compounds have a pyranose-pipecolinyl region, which is similar to leucine-proline peptide in the structure, and exhibit a variety of physiological activities by binding of peptidyl prolyl cis/trans isomerase.
The biosynthesis of polyketides is catalyzed by a megasynthase called polyketide synthase (PKS). These PKS complexes consist of several modules (sets of catalytic domains, each domain is responsible for one condensation step) involved in repetitive Claisen condensations from acyl-CoA, and the typical modular PKS is composed of a loading module, multiple extender modules, and a releasing module. The variation of catalytic domains within the modules affords the structural diversity in the resultant polyketide products with one-to-one correspondence. Among the modules, each extender module contains acyl transferase (AT), acyl carrier protein (ACP) and β-ketoacyl synthase (KS) domains that are directly involved in condensation of carboxylic acids, and also contains keto reductase (KR), dehydratase (DH), and enoyl reductase (ER) domains that are involved in reduction of a resultant β-keto group produced by the condensation. The addition of acyl-coA residue is mediated by one action of a module, in which AT transfers an acyl moiety to the corresponding ACP to produce acyl-ACP, and KS catalyzes the carbon-carbon bond formation via the Claisen condensation reaction of acyl group of acyl-ACP produced by the previous module, leading to an increase in the number of carbon. For instance, an action of this set mediates the addition of one acyl residue, resulting in an extension by two carbon atoms in the carbon chain backbone. During this process, KR, DH, and ER act in turn and thus, the β-keto group may be converted to an alcohol group, the alcohol group to a double bond, and the double bond to a saturated single bond.
Tacrolimus is a macrolide antibiotic having immunosuppressive properties, discovered from the culture broth of Streptomyces tsukubaensis, which is a bacterium found in the soil near Tsukuba, Northern Japan. Tacrolimus is an immunosuppressive agent used for the prevention of organ rejection after kidney and liver transplantation, approved by the US FDA in 1993, and is commercially available under the trade name of PROGRAF® (oral capsule or injectable) provided by Fujisawa Healthcare Inc. in 1994.
It was reported that tacrolimus is produced from strains such as Streptomyces tsukubaensis No. 9993 (U.S. Pat. No. 4,894,366), Streptomyces sp. ATCC55098, Streptomyces sp. MA 6858 (U.S. Pat. No. 5,116,756), Streptomyces sp. ATCC 53770, Streptomyces clavuligerus CKD1119 (Korean Patent No. 10-0485877), Streptomyces kanamyceticus KCC S-043 (KCTC 9225) or the like (Muramatsu H. et al., Actinomycetologica 19, 33-39 (2005)).
From a biosynthetic viewpoint, FK506 has a structurally unique feature in comparison with FK520 and rapamycin. FK506 is the only polyketide which carries an allyl side chain. Whereas the complete sequencing and characterization of the biosynthetic gene clusters of FK506 and FK520 were reported by Motamedi et al., (Eur. J. Biochem. 244, 528-34, 1998) and Wu et al., (Gene, 251, 81-90, 2000), respectively, only the partial sequence of FK506 gene cluster has been reported until quite recently. Consequently, the biosynthetic mechanism behind the introduction of the allyl functional group unique to FK506 has remained unresolved. The above biosynthetic pathway has to be identified first to prepare tacrolimus analogues with similar efficacy but reduced toxicity compared to tacrolimus or a tacrolimus analogue with the improved efficacy.
As FK506 analogues, FK520, dihydrotacrolimus, FK523, FK525 or the like are known, in which FK520 is a 23-membered macrolide compound and an ethyl analog of FK506 (Hatanaka H. et al., 1998), dihydrotacrolimus is a C21-propyl analogue of FK506, FK523 is a C21-methyl analogue of FK506, and FK525 is a prolyl analogue of FK506.